Extracellular serine protease cascades modulate protective responses to bleeding and infection in both mammals and arthropods. Experiments in this proposal are designed to investigate the roles of plasma serine proteases in innate immune responses, including activation of phenoloxidase, synthesis of antimicrobial proteins, and adhesion of hemocytes in a lepidopteran insect, Manduca sexta. The work will test a hypothesis that an initial interaction of a serine protease zymogen with one of several pattern recognition proteins in plasma bound to a pathogen surface triggers a conformational change that activates the protease and triggers a protease cascade in which the final protease in the pathway activates prophenoloxidase or a cytokine-like protein. Such immune responses are likely to affect the outcome of infections of insect vectors with the pathogens and parasites they transmit. The long-term goals of the research are to gain a thorough understanding of the protease cascades that mediate innate immune responses in M. sexta, to apply this knowledge to insect vectors of human diseases, and to apply advantages of the M. sexta system for fundamental studies on the regulation of serine protease activity. The specific aims of the project are: 1. Investigate the regulation of expression and activity of two plasma serine proteases: Pro-phenoloxidase activating proteinase activator and pro-plasmatocyte spreading peptide activator. 2. Identify proteases that activate signaling molecules, which stimulate expression of antimicrobial genes in fat body. 3. Identify proteases that can self-activate in the presence of a pattern recognition protein and a microbial polysaccharide, and begin to characterize the resulting proteinase cascade pathways. 4. Analyze the regulation of plasma proteinases by M. sexta serine protease inhibitors (serpins).